Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

BMC Microbiology

Table 3 Sequences of oligos used for amplification in qRT-PCR

Oligo Name	Oligo Sequence	mRNA/cDNA section amplified (bp from ATG)	Total length of mRNA (bp)
Igl 5' F	GCTGTTCCACATTGTGCATCAGTTTCAAATG	85–450 (Igl1), 85–459 (Igl2)	3306 (Igl1), 3318 (Igl2)
Igl 5' R	TTCTGCATGATCTTCTGTAGTTGCATTATCACATAAC
Igl 3' F	TGAAGGCACTTCTACAGAAGATAATAAAAT	2967–3166 (Igl1), 2979–3178 (Igl2)
Igl 3' R	TATGTCTTGAACATGGAATACATGATC
Igl1 F	TCTTGTAATAAGTTCCCGGAGCA	634–841 (Igl1)
Igl1 R	CATCAGAAACAGTACATCTTTTATTACATG
Igl2 F	GTACTAAATACCCAGATCATTGTTCAAA	643–841 (Igl2)
Igl2 R	CATCAGAAACAGTACATCTTTTATTACATG
URE3-BP 5' F	CCTGTAGCTAATTTCTGTTTATGGAATC	10–155	663
URE3-BP 5' R	CTTGTATATTGATCTAATGGGATAGTGTTAAG
URE3-BP Middle F	GATGAGAATTTTTGATACTGATTTTAATGGAC	276–454
URE3-BP Middle R	GATTAATATAGAATCCAAGTTGTTGAAGAG
URE3-BP 3' F	CTGTGATCTTAATTGTTGGATTG	504–658
URE3-BP 3' R	CCAAGAGGGAAGTAACAACGT
Actin F	GCACTTGTTGTAGATAATGGATCAGGAATG	variable (detects all family members/alleles)	variable
Actin R	ACCCATACCAGCCATAACTGAAACG
Jacob F	CAAAGGAGTTCAAATGGGATGTGTTAG	variable (detects all family members/alleles)	variable
Jacob R	TTATTTGGTGTAGGAGTTGGTAATGGG

Oligo pairs were designed to amplify short sections of Igl or URE3-BP. For Igl, four pairs of oligos were used: one amplifying the 5' end (Igl 5' oligo pair) and one the 3' end (Igl 3' oligo pair) of Igl1 and Igl2 simultaneously; and a pair each to amplify a short section unique to Igl1 or Igl2 (Igl1 oligo pair and Igl2 oligo pair, which have the same reverse primer in common) near the 5' end of the mRNA. Three oligo pairs were used to amplify short sections of URE3-BP: one pair the 5' end, one pair the middle, and one pair the 3' end. The actin and Jacob primers were designed to amplify all family members or alleles [35].

ISSN: 1471-2180